CN110603322A - Thermostable glucoamylases and methods of use thereof - Google Patents

Abstract

Polypeptides having glucoamylase activity, compositions comprising such polypeptides, and methods of using such polypeptides and compositions are disclosed.

Description

Thermostable glucoamylase and method of use

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Patent Application No. PCT/CN2013/076419, filed on March 07, 2017, which is incorporated herein by reference in its entirety.

technical field

The present disclosure relates to polypeptides having glucoamylase activity and compositions comprising such polypeptides. The present disclosure further relates to polynucleotides encoding such polypeptides, vectors and host cells comprising genes encoding such polypeptides, which vectors and host cells are also capable of producing such polypeptides. The present disclosure also relates to methods of saccharifying starch-containing materials using or applying the polypeptides or compositions, and saccharides produced by the methods. Furthermore, the present disclosure relates to methods of producing fermentation products and fermentation products produced by the methods.

Background technique

Glucoamylases (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) are enzymes that catalyze the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts.

The main application of glucoamylase is the saccharification of partially processed starch/dextrins to glucose, an essential substrate for many fermentation processes. The glucose can then be converted directly or indirectly into fermentation products using the fermenting organism. Examples of commercial fermentation products include alcohols (eg, ethanol, methanol, butanol, 1,3-propanediol); organic acids (eg, citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconates) , lactic acid, succinic acid, 2,5-diketo-D-gluconic acid); ketones (eg, acetone); amino acids (eg, glutamic acid); gases (eg, H ₂ and CO ₂ ); and More complex compounds include, for example, antibiotics (eg, penicillin and tetracycline); enzymes; vitamins (eg, riboflavin, _B12 , beta-carotene); hormones, and other compounds that are difficult to produce synthetically. Fermentation processes are also commonly used in the consumer alcohol (eg, beer and liquor), dairy products (eg, for the production of yogurt and cheese), leather, beverage, and tobacco industries.

The final product can also be a syrup. For example, the end product can be glucose, but also can be converted into fructose or a mixture consisting of nearly equal glucose and fructose, eg by glucose isomerase. This mixture, or a mixture further enriched in fructose, is the most commonly used high fructose corn syrup (HFCS) commercially worldwide.

Although a variety of microorganisms have been reported to produce glucoamylases (because they secrete large amounts of the enzyme extracellularly), glucoamylases for commercial purposes have traditionally been produced using filamentous fungi. However, commercially used fungal glucoamylases have certain limiting factors, such as moderate thermal stability, acidic pH requirements, and slow catalytic activity, which add to the cost of the process. Therefore, there is a need to find new glucoamylases to improve the temperature optimum, thereby improving the catalytic efficiency to shorten the saccharification time or obtain a higher yield of the final product.

It is an object of the present disclosure to provide certain polypeptides having glucoamylase activity, polynucleotides encoding the polypeptides, nucleic acid constructs useful for producing such polypeptides, compositions comprising the same, and uses of such polypeptides methods for different industrial applications.

SUMMARY OF THE INVENTION

Polypeptides, compositions and methods of saccharifying starch-containing materials using or applying the polypeptides or compositions of the present invention. Aspects and examples of such polypeptides, compositions and methods are described in the following individually numbered paragraphs.

1. In one aspect, a polypeptide having glucoamylase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence that is at least 95%, eg even at least 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 3;

(b) a polypeptide encoded by a polynucleotide under at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency conditions, and Even most preferably at least under very high stringency conditions hybridizes to (i) the mature polypeptide coding sequence of SEQ ID NO: 1,

(ii) a genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or

(iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 75%, more preferably at least 80%, of the mature polypeptide coding sequence of SEQ ID NO: 1 preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, even most preferably at least 95%, such as even at least 96%, 97% %, 98%, 99% or 100% identical nucleotide sequences;

(d) variants comprising one or more (eg, several) amino acid substitutions, deletions, and/or insertions of the mature polypeptide of SEQ ID NO: 2; and

(e) A fragment of the polypeptide of (a), (b), (c) or (d) having glucoamylase activity.

2. In another aspect, a polynucleotide comprising a nucleotide sequence encoding the polypeptide of paragraph 1.

3. In some embodiments of the polynucleotide of paragraph 2, the polynucleotide is operably linked to one or more control sequences that control production of the polypeptide within an expression host.

4. In another aspect, a recombinant host cell comprising the polynucleotide of paragraph 2.

5. In some embodiments of the host cell of paragraph 4, the host cell is an ethanologenic microorganism.

6. In some embodiments of the host cell of paragraph 4 or 5, the host cell further expresses and secretes one or more additional enzymes selected from the group consisting of protease, hemicellulase , cellulase, peroxidase, lipolytic enzyme, metal lipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase , mannanase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, lignase, alpha-amylase, pullulanase, phytase, tannase, pentosanase, Malanase, beta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, transferase, or a combination thereof.

7. On the other hand, a method for saccharifying a starch-containing material, the method comprising the steps of: i) contacting the starch-containing material with alpha-amylase; and ii) making the starch-containing material and glucose The glycoamylase is contacted at a temperature of at least 70°C; wherein the method produces at least 70% free glucose from the starch-containing material (substrate).

8. In some embodiments of the method of paragraph 7, wherein step (ii) is performed at a temperature of at least 75°C, preferably at least 80°C, for between 12 and 96 hours, preferably 18 to 60 hours.

9. In some embodiments of the method of paragraph 7 or 8, wherein the glucoamylase is at a temperature of at least 70°C, and/or a pH between 2.0 and 7.0, preferably between pH 4.0 and pH At least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% relative activity is maintained between pH 6.0, more preferably between pH 4.5 and pH 5.5.

10. In some embodiments of the method of any of paragraphs 7-9, wherein the method comprises performing step (i) and step (ii) sequentially or simultaneously.

11. In some embodiments of the method of any of paragraphs 7-10, wherein the method further comprises pre-saccharification prior to saccharification step ii).

12. In some embodiments of the method of any of paragraphs 7-11, wherein the glucoamylase is the polypeptide of claim 1.

13. In some embodiments of the method of any of paragraphs 7-12, wherein step (i) is performed at or below the gelatinization temperature of the starch-containing material.

14. In some embodiments of the method of any of paragraphs 7-13, wherein no additional debranching enzymes are present in step (i) and/or step (ii).

15. In some embodiments of the method of paragraph 14, wherein the debranching enzyme is a pullulanase.

16. In another aspect, a sugar produced by the method of any of paragraphs 7-15.

17. In another aspect, a method of producing a fermentation product from the sugar of paragraph 16, wherein the sugar is fermented by a fermenting organism.

18. In some embodiments of the method of paragraph 17, wherein the fermentation process is performed sequentially or concurrently with step (ii).

19. In some embodiments of the method of paragraph 17 or 18, wherein the fermentation product comprises ethanol.

20. In some embodiments of the method of paragraph 17 or 18, wherein the fermentation product comprises a non-ethanol metabolite.

21. In some embodiments of the method of paragraph 20, wherein the metabolite is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, glucose Potassium acid, organic acid, glucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, isobutanol, amino acid, lysine, tyrosine, threonine, glycinol, itaconic acid, 1 , 3-Propanediol, vitamins, enzymes, hormones, isoprene, or other biochemicals or biological materials.

22. In another aspect, a method of applying the polypeptide of paragraph 1 in brewing.

Description of drawings

Figure 1 is a plasmid map of pJG580.

Figure 2 is a graph of the product distribution of PruGA1 fermented for 95 hours.

Figure 3 is a comparison of DP1 production of PruGA1-0.3x, PruGA1-1x, AfuGA-1x, and AnGA-1x at 60°C, 65°C and 70°C after 72-h incubation.

Figure 4 is a comparison of the activity of PruGA1 and TrGA on crude starch at pH 3.5.

Figure 5 is a comparison of the activity of PruGA1 and TrGA on crude starch at pH 4.5.

Detailed ways

The present disclosure relates to polypeptides having glucoamylase activity and compositions comprising such polypeptides. The present disclosure further relates to polynucleotides encoding such polypeptides, vectors and host cells comprising genes encoding such polypeptides, which vectors and host cells are also capable of producing such polypeptides. The present disclosure also relates to methods of saccharifying starch-containing materials using or applying the polypeptides or compositions, and saccharides produced by the methods. Furthermore, the present disclosure relates to methods of producing fermentation products and fermentation products produced by the methods.

Before describing the compositions and methods in detail, the following terms and abbreviations are defined.

Unless otherwise defined, all technical and scientific terms used have their ordinary meaning in the relevant scientific field. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, John Wiley and Sons, New York (1994), and Hale and Markham, Harper The Collins Dictionary of Biology, Harper Perennial, NY (1991) provides the ordinary meanings of many of the terms describing the present invention.

definition

The term "glucoamylase (1,4-α-D-glucanoglucohydrolase, EC 3.2.1.3) activity" is defined herein as catalyzing the release from the non-reducing end of starch or related oligosaccharide and polysaccharide molecules Enzymatic activity of D-glucose.

Polypeptides of the invention have at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the mature polypeptide of SEQ ID NO: 2 glycoamylase activity.

The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein" and "peptide" and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". Amino acid sequences are expressed in standard amino-to-carboxy-terminal orientation (ie, N→C) using conventional one-letter or three-letter codes for amino acid residues.

The term "mature polypeptide" is defined herein as a polypeptide in its final form after translation and any post-translational modifications (eg, N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one aspect, the mature polypeptide is SEQ ID NO:2 based on the SignalP (Nielsen et al., 1997, Protein Engineering 10:1-6) program that predicts amino acids 1 to 21 of SEQ ID NO:2 to be a signal peptide of amino acids 22 to 614.

The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded, and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences encoding particular amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

The term "coding sequence" means a nucleotide sequence that directly specifies the amino acid sequence of a protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with an ATG start codon or alternative start codons (eg, GTG and TTG), and ends with a stop codon (eg, TAA, TAG, and TGA) Finish. Coding sequences can be DNA, cDNA, synthetic or recombinant nucleotide sequences.

The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription of mature spliced mRNA molecules obtained from eukaryotic cells. cDNA lacks intronic sequences that can be present in the corresponding genomic DNA. Early primary RNA transcripts are precursors to mRNAs that undergo a series of steps before being presented as mature spliced mRNAs. These steps include the removal of intronic sequences through a process called splicing. Therefore, cDNA derived from mRNA lacks any intronic sequences.

The term "hybridization" refers to the process by which a nucleic acid strand forms a duplex (ie, base pairing) with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization at 65°C and 0.1X SSC (where 1X SSC = 0.15M NaCl, 0.015M trisodium citrate, pH 7.0). Hybridized double-stranded nucleic acids are characterized by a melting temperature ( _Tm ) in which half of the hybridized nucleic acids do not pair with the complementary strand. Mismatched nucleotides within the duplex lower the _Tm .

"Synthetic" molecules are produced by chemical or enzymatic synthesis in vitro rather than by an organism.

A "host strain" or "host cell" is an organism into which an expression vector, phage, virus or other DNA construct has been introduced, including a polynucleotide encoding a polypeptide of interest (eg, an amylase). Exemplary host strains are microbial cells (eg, bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and/or fermenting sugars. The term "host cell" includes protoplasts produced from the cell.

The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

The term "vector" refers to a polynucleotide sequence designed to introduce nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

An "expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest operably linked to suitable control sequences capable of effecting DNA expression in a suitable host. Such control sequences may include promoters that affect transcription, optional operator sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

The term "control sequences" is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or each control sequence may be native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with a plurality of linkers for the purpose of introducing specific restriction sites that facilitate ligation of the control sequences to the coding region of the nucleotide encoding the polypeptide.

The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates extracellular secretion of the protein. The mature form of the extracellular protein lacks the signal sequence that is cleaved during secretion.

"Biologically active" refers to a sequence having a specified biological activity (eg, enzymatic activity).

The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is usually expressed as units (U)/mg protein.

"Percent sequence identity" means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a given reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. [Nucleic Acids Research] 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

The term "homologous sequence" is defined herein as a tfasty search with the Penicillium russellii glucoamylase of SEQ ID NO: 3 (Pearson, W.R., 1999, in Bioinformatics Methods and Protocols [Bioinformatics Methods] and Protocol], S. Misener and S.A. Krawetz, editors, pp. 185-219) for predicted proteins with E-values (or expected scores) less than 0.001.

The term "polypeptide fragment" is defined herein as a polypeptide having one or more (eg, several) amino acids deleted from the amino and/or carboxyl terminus of SEQ ID NO: 3, or a homologous sequence thereof, wherein the fragment has Glucoamylase activity.

With respect to a polypeptide, the terms "wild-type," "parent," or "reference" refer to a naturally-occurring polypeptide that does not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, with respect to a polynucleotide, the terms "wild-type", "parental" or "reference" refer to a naturally occurring polynucleotide that does not contain artificial nucleoside changes. Note, however, that polynucleotides encoding wild-type, parental, or reference polypeptides are not limited to naturally occurring polynucleotides, and encompass any polynucleotides encoding wild-type, parental, or reference polypeptides.

The terms "thermostable" and "thermostability" in reference to an enzyme refer to the ability of an enzyme to remain active after exposure to elevated temperatures. The thermostability of an enzyme (eg amylase) is measured by its half-life (t _1/2 ), given in minutes, hours or days, during which half of the enzyme activity is lost under defined conditions. Half-life can be calculated, for example, by measuring residual alpha-amylase activity after exposure (ie, challenged) to elevated temperatures.

A "pH range" in reference to an enzyme refers to the range of pH values at which the enzyme exhibits catalytic activity.

The terms "pH stability" and "pH stability" in reference to an enzyme relate to the ability of an enzyme to maintain activity for a predetermined period of time (eg, 15 min, 30 min, 1 h) at a wide range of pH values.

The term "pre-saccharification" is defined herein as a process prior to full saccharification or simultaneous saccharification and fermentation (SSF). Pre-saccharification is usually carried out at a temperature of about 60°C for 40-90 minutes between 30°C and 65°C.

The phrase "simultaneous saccharification and fermentation (SSF)" refers to a biochemical production process in which a microorganism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present in the same process step. SSF involves the simultaneous hydrolysis of starch substrates (granulated, liquefied or dissolved) to sugars (including glucose) and the fermentation of sugars to alcohols or other biochemicals or biological materials in the same reaction vessel.

"Slurry" is an aqueous mixture containing insoluble starch granules in water.

The term "total sugar content" refers to the total soluble sugar content present in a starch composition including monosaccharides, oligosaccharides and polysaccharides.

The term "dry solids" (ds) refers to dry solids dissolved in water, dry solids dispersed in water, or a combination of the two. Dry solids thus include granular starch and its hydrolyzates, including glucose.

"Dry solids" content refers to the percentage by weight of dissolved and dispersed dry solids relative to the water in which the dry solids are dispersed and/or dissolved. The initial dry solids content of the starch is the weight of the granular starch calculated as water content divided by the weight of the granular starch plus the weight of water. Subsequent dry solids levels can be determined from the initial levels adjusted for any added or lost water and chemical gain. The subsequent dissolved dry solids content can be measured from the refractive index as shown below. 8

The term "high DS" refers to an aqueous starch slurry having a dry solids content greater than 38% (wt/wt).

"Dry matter starch" refers to the dry starch content of a substrate, eg, starch slurry, and can be determined by subtracting the contribution of any non-starch components, such as protein, fiber, and water, from the mass of the substrate. For example, if the granular starch slurry has a water content of 20% (wt/wt) and a protein content of 1% (wt/wt), then 100 kg of granular starch has a dry starch content of 79 kg. Dry matter starch can be used to determine the number of enzyme units to use.

"Degree of Polymerization (DP)" refers to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP4+ (>DP3) represents a polymer with a degree of polymerization greater than 3.

The term "contacting" refers to placing reference components (including, but not limited to, enzymes, substrates, and fermenting organisms) in sufficient proximity to affect a desired result, eg, the action of the enzyme on the substrate or the fermenting organism fermenting the substrate. Those skilled in the art will recognize that mixing solutions can cause "contacting". "Ethanologenic microorganism" refers to a microorganism that has the ability to convert sugars or other sugars to ethanol.

The term "biochemical" refers to microbial metabolites such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, delta-gluconate Esters, sodium erythorbate, omega 3 fatty acids, butanol, isobutanol, amino acids, lysine, itaconic acid, other organic acids, 1,3-propanediol, vitamins, or isoprene, or other biological materials.

The term "pullulanase" is also known as a debranching enzyme (E.C. 3.2.1.41, amylopectin 6-glucanohydrolase), capable of hydrolyzing alpha-1-6-glycosidic bonds in amylopectin molecules.

The term "about" refers to ±15% of the reference value.

The term "comprising" and its cognates are used in its inclusive sense; ie, equivalent to the term "comprising" and its corresponding cognates.

EC Enzyme Committee

CAZy Carbohydrate Active Enzyme

w/v weight/volume

w/w weight/weight

v/v volume/volume

wt% weight percentage

°C Celsius

g or gm grams

μg microgram

mg mg

kg kilogram

μL and μl microliters

ml and mL milliliters

mm mm

μm micrometer

mole mole

mmol mmol

M mole

mM Millimoles

μM micromolar

nm nanometer

U unit

ppm parts per million

hr and h hours

EtOH Ethanol

ds dry solids

Polypeptides with glucoamylase activity

In a first aspect, the present invention relates to a polypeptide comprising an amino acid sequence which is preferably at least 90%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 93% of the polypeptide of SEQ ID NO:3 Preferably at least 94%, and even most preferably at least 95%, such as at least 96%, 97%, 98%, 99% or 100% identical, the polypeptide has glucoamylase activity.

In some embodiments, the polypeptide of the present invention is a homologous polypeptide comprising an amino acid sequence that differs from the polypeptide of SEQ ID NO: 3 by ten amino acids, preferably by nine amino acids, preferably by eight amino acids , preferably differ by seven amino acids, preferably differ by six amino acids, preferably differ by five amino acids, more preferably differ by four amino acids, even more preferably differ by three amino acids, most preferably differ by two amino acids, and even most It preferably differs by one amino acid.

In some embodiments, the polypeptide of the invention is a variant of the polypeptide of SEQ ID NO: 3, or a fragment thereof having glucoamylase activity.

In some embodiments, the polypeptides of the invention are thermostable and retain glucoamylase activity at elevated temperatures. Polypeptides of the invention exhibit thermal stability at pH values ranging from about 2.5 to about 8.0 (eg, about 3.0 to about 7.5, about 3.0 to about 7.0, about 3.0 to about 6.5, etc.). For example, at a pH of from about 3.0 to about 7.0 (eg, from about 3.5 to about 6.5, etc.), the polypeptides of the invention are at high temperature (eg, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C or higher) to maintain most of the glucoamylase activity for an extended period of time (e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 5 hours, or even longer). For example, a polypeptide of the invention retains at least about 35% (eg, at least 35%) when incubated at an increased temperature at a pH of from about 3.5 to about 6.5 for at least 1 hour, 3 hours, 5 hours, or even longer. about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% or higher percentages) of glucoamylase activity .

In some embodiments, at a temperature of 50°C, the polypeptides of the invention have maximal activity at about pH 5, and 90% of maximal activity at pH about 3.5 to about pH 6.0, as measured by the assays described herein above, and more than 70% of maximum activity at pH about 2.8 to pH about 7.0. Exemplary pH ranges for use with enzymes are pH 2.5-7.0, 3.0-7.0, 3.5-7.0, 2.5-6.0, 3.0-6.0, 3.5-6.0.

In some embodiments, at a pH of 5.0, the polypeptides of the invention have maximum activity at a temperature of about 75°C, at a temperature of about 63°C to a temperature of about 79°C, as measured by the assays described herein It has more than 70% of the maximum activity. Exemplary temperature ranges for using enzymes are 50°C-82°C, 50°C-80°C, 55°C-82°C, 55°C-80°C, and 60°C-80°C. In a second aspect, the present invention relates to a polypeptide having glucoamylase activity encoded by a polynucleotide under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, More preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the mature polypeptide comprising SEQ ID NO: 1 The genomic DNA sequence of the polypeptide coding sequence, or the full-length complementary strand hybridization of (iii)(i) or (ii) (J.Sambrook, E.F.Fritsch and T.Maniatis, 1989, Molecular Cloning, A Laboratory Manual] , 2nd ed., Cold Spring Harbor [Cold Spring Harbor Laboratory], New York).

Nucleic acid probes can be designed using the nucleotide sequence of SEQ ID NO: 1; or fragments thereof to identify and clone against strains from different genera or species encoding glucoamylase activity according to methods well known in the art of polypeptide DNA. In particular, such probes can be used to hybridize to the genome or cDNA of a genus or species of interest according to standard Southern blotting procedures in order to identify and isolate the corresponding genes therein. Such probes can be much shorter than the complete sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. However, it is preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides in length, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes can be used, for example preferably at least 600 nucleotides in length, more preferably at least 800 nucleotides, even more preferably at least 1000 nucleotides, even more preferably at least 1500 nucleotides in length nucleotides, or most preferably nucleic acid probes of at least 1800 nucleotides. Both DNA and RNA probes can be used. Probes are typically labeled (eg, ^with32P , ^3H , ^35S , biotin, or avidin) to detect the corresponding gene. The present invention also encompasses such probes.

Thus, genomic DNA or cDNA libraries prepared from such other strains can be screened for DNA that hybridizes to the probes described above and encodes a polypeptide having glucoamylase activity. Genomic or other DNA from such other strains can be isolated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA or isolated DNA from the library can be transferred and immobilized on nitrocellulose or other suitable support material. In order to identify clones or DNAs homologous to SEQ ID NO: 1 or a subsequence thereof, carrier materials are preferably used in Southern blotting.

In a third aspect, the present invention relates to a polypeptide having glucoamylase activity encoded by a polynucleotide comprising, preferably at least 60%, more preferably at least 60%, more preferably a at least 63%, more preferably at least 65%, more preferably at least 68%, more preferably at least 70%, more preferably at least 72%, more preferably at least 75%, at least 77%, more preferably at least 79%, more preferably at least 81%, more preferably at least 83%, more preferably at least 85%, more preferably at least 90%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, And even most preferably a degree of sequence identity of at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity, the polynucleotide encodes a polypeptide having glucoamylase activity.

In a fourth aspect, the glucoamylases of the invention comprise conservative substitutions of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO:3. Exemplary conservative amino acid substitutions are listed in Table 1. Some conservative mutations can be created by genetic manipulation, while others are created by introducing synthetic amino acids into the polypeptide by other means.

Table 1. Conservative amino acid substitutions

In some embodiments, the glucoamylases of the invention comprise deletions, substitutions, insertions or additions of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO: 3 or a homologous sequence thereof. In some embodiments, the glucoamylases of the invention are derived from the amino acid sequence of SEQ ID NO:3 by conservative substitution of one or several amino acid residues. In some embodiments, the glucoamylases of the invention are derived from the amino acid sequence of SEQ ID NO:3 by deletion, substitution, insertion, or addition of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO:3 . In all cases the expression "one or several amino acid residues" means 10 or less, ie 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

Alternatively, the amino acid change is of such a nature that it alters the physicochemical properties of the polypeptide. For example, amino acid changes can increase the thermal stability of the polypeptide, alter substrate specificity, alter pH optimum, and the like.

Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known mutagenesis, recombination and/or shuffling methods followed by a relevant screening program such as that described by Reidhaar- Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; Those disclosed in WO 95/22625. Other methods that can be used include error-prone PCR, phage display (eg, Lowman et al., 1991, Biochem. 30:10832-10837; US Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire) et al, 1986, Gene 46:145; Ner et al, 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest and can be applied to polypeptides of unknown structure.

The amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO:2 may be at most 10, preferably at most 9, more preferably at most 8, more preferably at most 7, more preferably at most 6, More preferably at most 5, more preferably at most 4, even more preferably at most 3, most preferably at most 2, and even most preferably at most 1.

The glucoamylase can be a "chimeric" or "hybrid" polypeptide in that it includes at least a portion from a first glucoamylase, and from a second amylase, glucoamylase, beta-amylase, alpha- Glucosidases or at least a portion of other starch-degrading enzymes, or even other glycosyl hydrolases, such as, but not limited to, cellulases, hemicellulases, etc. (including chimeras recently "rediscovered" as domain-exchanged amylases amylase). The glucoamylases of the present invention may further comprise a heterologous signal sequence, ie an epitope that allows tracking or purification and the like.

Production of glucoamylase

The glucoamylases of the invention can be produced in host cells, eg, by secretion or intracellular expression. Following secretion of the glucoamylase into the cell culture medium, cultured cell material (eg, whole cell broth) containing the glucoamylase can be obtained. Optionally, the glucoamylase can be isolated from the host cells, or even from the cell culture fluid, depending on the desired purity of the final glucoamylase. The gene encoding the glucoamylase can be cloned and expressed according to methods well known in the art. Suitable host cells include bacteria, fungi (including yeast and filamentous fungi) and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae, Trichoderma reesi, or Myceliopthora Thermophila. Other host cells include bacterial cells such as Bacillus subtilis or B. licheniformis, and Streptomyces.

Additionally, the host may express one or more coenzymes, proteins, peptides. These can benefit liquefaction, saccharification, fermentation, SSF and downstream processing. In addition, host cells can produce ethanol and other biochemicals or biological materials in addition to the enzymes used to digest various feedstocks. Such host cells can be used in fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need for added enzymes.

combination

The present invention also relates to compositions comprising the polypeptides of the present invention. In some embodiments, an amino acid sequence comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 can also be included The polypeptides are used in enzyme compositions. Preferably, these compositions are formulated to provide desirable characteristics such as light color, low odor, and acceptable storage stability.

These compositions may contain the polypeptides of the invention as the main enzymatic component, eg, one-component compositions. Alternatively, the composition may comprise multiple enzymatic activities such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, keratinase, cyclodextrin glycosyl Transferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, beta-amylase, isoamylase, haloperoxidase enzyme, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme , pullulanase, ribonuclease, transglutaminase, xylanase, or a combination thereof, the enzymes may be added in effective amounts well known to those skilled in the art.

Polypeptide compositions can be prepared according to methods known in the art, and these polypeptide compositions can be in the form of liquid or dry compositions. For example, a composition comprising a glucoamylase of the present invention may be an aqueous or non-aqueous formulation, granules, powders, gels, slurries, pastes, etc., which may further comprise the additional enzymes listed herein of any one or more, as well as buffers, salts, preservatives, water, co-solvents, surfactants, and the like. Such compositions may be combined with endogenous enzymes or other ingredients already present in slurries, water baths, washing machines, food or beverage products, etc. (eg, endogenous plant (including algal) enzymes, residual enzymes from previous processing steps, etc.) Combination works. The polypeptides included in the composition can be stabilized according to methods known in the art.

The composition may be a cell expressing the polypeptide, including cells capable of producing a product from fermentation. Such cells can be provided in cream or dry form with suitable stabilizers. Such cells may further express additional polypeptides, such as those mentioned above.

Examples of preferred uses of the polypeptides or compositions of the invention are given below. Dosages of the polypeptide compositions of the present invention, as well as other conditions for using the compositions, can be determined based on methods known in the art.

The above compositions are suitable for use in liquefaction, saccharification, and/or fermentation processes, preferably in starch conversion, especially for the production of syrups and fermentation products such as ethanol.

use

The present invention is also directed to the use of the polypeptides or compositions of the present invention in liquefaction processes, saccharification processes, and/or fermentation processes. The polypeptide or composition can be used in a single process, such as in a liquefaction process, a saccharification process, or a fermentation process. The polypeptide or composition may also be used in a combination of processes (eg, in a liquefaction and saccharification process, in a liquefaction and fermentation process, or in a saccharification and fermentation process (preferably in relation to starch conversion)).

1. Liquefaction

As used herein, the term "liquefaction or liquefy" means a method of converting gelatinized starch into a lower viscosity liquid containing shorter chain soluble dextrins, optionally with the addition of liquefaction-inducing enzymes and/or saccharification enzymes. In some embodiments, the prepared starch substrate is slurried with water. The starch slurry may contain starch at a dry solids weight percentage of about 10%-55%, about 20%-45%, about 30%-45%, about 30%-40%, or about 30%-35%. For example, alpha-amylase (EC 3.2.1.1) can be added to the slurry using a metering pump. Alpha-amylases commonly used for this application are thermostable bacterial alpha-amylases, such as Geobacillus stearothermophilus alpha-amylase, Cytophaga alpha-amylase, etc., such as Spezyme (DuPont product), Spezyme AA (DuPont product), Fred (DuPont), Clearflow AA (DuPont), Spezyme Alpha PF (DuPont), Spezyme Powerliq (DuPont) can all be used herein.

The starch plus alpha-amylase slurry can be pumped continuously through a jet cooker (which is steam heated to 80°C-110°C, depending on the source of the starch-containing feedstock). Under these conditions, gelatinization occurs rapidly and the enzymatic activity in combination with significant shear forces begins to hydrolyze the starch substrate. The residence time in the jet cooker is short. The partially gelatinized starch can then enter a series of retention tubes maintained at 105°C-110°C for 5-8 min to complete the gelatinization process ("primary liquefaction"). Hydrolysis for the desired DE is accomplished in a retention tank at 85°C-95°C or higher for about 1 to 2 hours ("secondary liquefaction"). The slurry was then cooled to room temperature. This cooling step can be from 30 minutes to 180 minutes, such as 90 minutes to 120 minutes. The liquefied starch is usually in the form of a slurry with a dry solids content (w/w) of about 10%-50%; about 10%-45%; about 15%-40%; about 20%-40%; about 25% -40%; or about 25%-35%.

In a conventional enzymatic liquefaction process, a thermostable alpha-amylase is added and the long chain starch is degraded into branched and linear shorter units (maltodextrins), but no glucoamylase is added. The glucoamylase of the present invention is highly thermostable, so it is advantageous to add the glucoamylase during the liquefaction process.

2. Saccharification

Alpha-amylase and glucoamylase (optionally in the presence of another enzyme or enzymes) can be used to saccharify the liquefied starch into a syrup rich in low DP (eg, DP1+DP2) sugars. The exact composition of the saccharified product depends on the combination of enzymes used and the type of starch being processed. Advantageously, the syrup obtainable using the provided glucoamylases may contain more than 30% by weight of DP2 of the total oligosaccharides in the saccharified starch, such as 45%-65% or 55%-65%. The weight percentage of (DP1+DP2) in the saccharified starch may exceed about 70%, eg, 75%-85% or 80%-85%.

Liquefaction is usually carried out as a continuous process, while saccharification is usually carried out as a batch process. Saccharification conditions depend on the nature of the liquefaction and the type of enzymes available. In some cases, the saccharification process can involve a temperature of about 60°C-65°C and a pH of about 4.0-4.5 (eg, pH 4.3). The saccharification can be carried out, for example, at a temperature of about 40°C, about 50°C, or about 55°C to about 60°C or about 65°C, it being necessary to cool the liquefaction. The pH can be adjusted as needed. Saccharification is usually done in a stirred tank, which can take hours to fill or empty. Enzymes are usually added to the dry solids at a fixed rate (when the tank is being filled), or in a single dose (at the beginning of the filling phase). The saccharification reaction to prepare the syrup is generally carried out for about 24-72 hours, eg, 24-48 hours. However, pre-saccharification, typically 40-90 minutes, is typically only performed at temperatures between 30°C-65°C (usually about 60°C), followed by full saccharification in simultaneous saccharification and fermentation (SSF). The glucoamylases of the present invention are highly thermostable, so the pre-saccharification and/or saccharification of the present invention can be carried out at higher temperatures than conventional pre-saccharification and/or saccharification. In one embodiment, the method of the present invention comprises pre-saccharification of the starch-containing material prior to a simultaneous saccharification and fermentation (SSF) process. Pre-saccharification can be performed at elevated temperature (eg, 50°C-85°C, preferably 60°C-75°C) prior to transfer into SSF. Preferably, saccharification is best performed at higher temperatures ranging from about 30°C to about 75°C (eg, 45°C-75°C or 50°C-75°C). By performing the saccharification process at a higher temperature, the process can be performed in a shorter period of time, or alternatively can be performed with a lower enzyme dosage. Furthermore, the risk of microbial contamination is reduced when the liquefaction and/or saccharification process is performed at higher temperatures.

In a preferred aspect of the present invention, the liquefaction and/or saccharification comprises sequential or simultaneous liquefaction and saccharification processes.

3. Fermentation

Soluble starch hydrolysates (particularly glucose-enriched syrups) can be fermented by contacting the starch hydrolysate with a fermenting organism, typically at a temperature of about 32°C (eg, from 30°C to 35°C). "Fermenting organism" refers to any organism (including bacterial and fungal organisms) suitable for use in a fermentation process and capable of producing a desired fermentation product. Particularly suitable fermenting organisms are capable of directly or indirectly fermenting (ie, converting) sugars (eg, glucose or maltose) into desired fermentation products. Examples of fermenting organisms include yeast (eg, Saccharomyces cerevisiae) and bacteria (eg, Zymomonas mobilis) that express alcohol dehydrogenase and pyruvate decarboxylase. Ethanologenic microorganisms can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. For example, improved strains of ethanologenic microorganisms that can withstand higher temperatures are known in the art and can be used. See Liu et al., (2011) ShengWu Gong Cheng Xue Bao [Journal of Bioengineering] 27:1049-56. Commercially available yeast include, for example, Red Star(TM)/Lesaffre Ethanol Red (available from Red Star/Lesaffre, USA), FALI (from Burns Phillips Foods, Inc., USA) Fleischmann's Yeast, a division of the company (Burns Philp Food Inc.), SUPERSTART (available from Alltech), GERT STRAND (available from Gert Strand AB, Sweden) ), and FERMIOL (available from DSM Specialties). The temperature and pH of the fermentation will depend on the fermenting organism. Microorganisms that produce other metabolites such as citric acid and lactic acid by fermentation are also known in the art. See, eg, Papagianni (2007) Biotechnol. Adv. [Advance in Biotechnology] 25:244-63; John et al. (2009) Biotechnol. Adv. [Advance in Biotechnology] 27:145-52.

The saccharification and fermentation process can be performed as an SSF process. The SSF process can be performed with fungal cells that continuously express and secrete glucoamylase throughout the SSF. A fungal cell expressing a glucoamylase can also be a fermenting microorganism, such as an ethanologenic microorganism. Thus, ethanol production can be carried out using fungal cells expressing sufficient glucoamylase to require less or no exogenous addition of the enzyme. Fungal host cells can be derived from appropriately engineered fungal strains. In addition to glucoamylases, fungal host cells that express and secrete other enzymes can also be used. Such cells can express amylase and/or pullulanase, phytase, alpha-glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, proteases, Beta-glucosidase, pectinase, esterase, oxidoreductase, transferase, or other enzymes. Ethanol can be recovered after fermentation.

4. Crude starch hydrolysis

The present invention provides the use of the glucoamylase of the present invention for producing glucose and the like from crude starch or granular starch. Generally, the glucoamylases of the present invention, alone or in the presence of an alpha-amylase, can be used in a crude starch hydrolysis (RSH) or granular starch hydrolysis (GSH) process for the production of desired sugars and fermentation products. The granular starch is dissolved by enzymatic hydrolysis below the gelatinization temperature. Such "low temperature" systems (also known as "no cooking" or "cold cooking") are reported to be capable of handling higher concentrations of dry solids (eg, up to 45%) than conventional systems.

The "Rough Starch Hydrolysis" process (RSH) differs from conventional starch processing processes and involves, typically in the presence of at least glucoamylase and/or amylase, at or below the gelatinization temperature of the starch substrate Sequential or simultaneous saccharification and fermentation of granular starch. Starches heated in water begin to gelatinize between 50°C and 75°C, the exact temperature of gelatinization depends on the particular starch. For example, the gelatinization temperature can vary depending on the plant species, the specific variety of plant species, and growth conditions. In the context of the present invention, the gelatinization temperature of starch is given by applying the method described by Gorinstein. The temperature at which the birefringence loss of the particles is 5%.

The glucoamylases of the present invention can also be used in combination with enzymes that hydrolyze only alpha-(1,6)-glycosidic bonds in molecules containing at least four glucose residues. Preferably, the glucoamylase of the present invention is used in combination with a pullulanase or an isoamylase. Isoamylase and pullulanase for starch debranching are described in G.M.A. van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, 101-142 Uses, molecular properties of enzymes, and potential uses of enzymes with glucoamylases.

In another aspect, the present invention relates to the use of a glucoamylase of the present invention, comprising converting starch into eg syrupy beverages and/or fermentation products (including ethanol).

5. Fermentation products

The term "fermentation product" means a product produced by a process including a fermentation process using a fermenting organism. Fermentation products contemplated according to the present invention include alcohols (eg, arabitol, butanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol , glycerin, sorbitol, and xylitol); organic acids (eg, acetic acid, acetic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid , fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); ketones (eg, acetone); amino acids (eg, aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); alkanes Hydrocarbons (eg, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (eg, cyclopentane, cyclohexane, cycloheptane, and cyclooctane); olefins (eg, pentene, hexene, heptene, and octene); gases (eg, methane, hydrogen ( _H2 ), carbon dioxide ( _CO2 ), and carbon monoxide (CO)) ; antibiotics (eg, penicillin and tetracycline); enzymes; vitamins (eg, riboflavin, _B12 , beta-carotene) and hormones. In preferred aspects, the fermentation product is ethanol, eg, fuel ethanol; potable ethanol, which is neutral alcohol ready to drink; or industrial ethanol or consumer alcohol industry (eg, beer and liquor), dairy industry (eg, fermented dairy products) , products used in the leather industry and the tobacco industry. Preferred types of beer include ale, stout, porter, lager, bitter, malt liquor, high alcohol beer, Low-alcohol beer, low-calorie beer, or light beer. Preferred fermentation processes for use include alcoholic fermentation processes well known in the art. Preferred fermentation methods are anaerobic fermentation methods, which are well known in the art.

6. Brewing

The glucoamylases of the present invention are highly thermostable, so they can be used for starch hydrolysis at high temperatures to prepare fermented malt beverages. For example, the glucoamylases of the present invention can be added to a hot mash, utilizing elevated temperature to increase the reaction rate and increase the yield of fermentable sugars prior to the addition of yeast. In combination with amylase and optional pullulanase and/or isoamylase, glucoamylase helps convert starch to dextrin and fermentable sugars, reducing residual non-fermentable carbohydrates in the final beer. The glucoamylases of the present invention are added in effective amounts that can be readily determined by those skilled in the art.

Processes for making beer are well known in the art. See eg Wolfgang Kunze (2004) "Technology Brewing and Malting" Research and Teaching Institute of Brewing, Berlin (VLB), 3rd ed. Briefly, the process involves: (a) making a mash, (b) filtering the mash to make wort, and (c) fermenting the wort to obtain a fermented beverage (eg, beer).

The brewing composition comprising glucoamylase in combination with amylase and optionally pullulanase and/or isoamylase may be added to the mash of step (a) above, i.e. during the preparation of the mash . Alternatively or additionally, the brewing composition may be added to the mash of step (b) above, ie during filtration of the mash. Alternatively or additionally, the brewing composition may be added to the wort of step (c) above, ie during fermentation of the wort.

All references cited herein are incorporated by reference in their entirety for all purposes. In order to further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative and not restrictive.

Example

Example 1

Sequence of Penicillium russellus glucoamylase (PruGA1)

A strain of Penicillium russellus available for industrial applications was selected as a potential source of various enzymes. The entire genome of the Penicillium russells strain was sequenced and the nucleotide sequence of a putative glucoamylase (designated "PruGA1") was determined by sequence identity. The gene encoding PruGA1 is shown in SEQ ID NO: 1:

The amino acid sequence of the PruGA1 precursor protein is shown in SEQ ID NO: 2:

The amino acid sequence of the mature form of PruGA1 confirmed by N-terminal Edman degradation is shown in SEQ ID NO:3:

Example 2

Expression and Purification of Penicillium Russell Glucoamylase (PruGA1)

The nucleotide sequence of the synthesized PruGA1 gene from Penicillium russellus is shown in SEQ ID NO: 4:

The DNA sequence of PruGA1 was optimized for expression of PruGA1 in Trichoderma reesei and inserted into the pTrex3gM expression vector (described in US Published Application 2011/0136197A1), resulting in pJG580 (Figure 1).

Plasmid pJG580 was transformed into a Trichoderma reesei strain (described in WO05/001036) using protoplast transformation (Te'o et al., J. Microbiol. Methods 51:393-99 2002). The transformants were selected and fermented by the method described in WO 2016/138315. Supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis and enzymatic activity assays.

A seed culture of the above transformed cells was then grown in defined medium in a 2.8 L fermentor. Fermentation broths were sampled for SDS-PAGE analysis at fermentation times of 42, 65 and 95 hours to measure dry cell weight, residual glucose and extracellular protein concentration. Figure 2 shows the product profile of PruGA1 fermentation at 95 hours. After 95 hours of fermentation, followed by centrifugation, filtration and concentration, a 500 mL concentrated sample was obtained. The protein concentration was determined to be 10.7 g/L by the BCA method.

200 mL of clarified broth was loaded onto a 20-mL β-cyclodextrin-coupled Sepharose 6 column (pre-equilibrated with 20 mM sodium acetate (pH 5.0), 150 mM NaCl) and washed with 3 column volumes of the same buffer . Elution was performed using 5 column volumes of 10 mM alpha-cyclodextrin in 20 mM sodium acetate (pH 5.0) containing 150 mM NaCl. Fractions were collected and assayed for glucoamylase activity and subjected to SDS-PAGE. Fractions containing the target protein were pooled and concentrated in an Amicon Ultra-15 apparatus with 10K MWCO using 20 mM sodium acetate (pH 5.0) containing 150 mM NaCl. Purified samples were greater than 99% pure and were stored in 40% glycerol at -80°C until use.

Example 3

Specific activity of PruGA1 on soluble starch

Glucoamylase was determined based on the release of glucose from soluble starch by glucoamylase using the coupled glucose oxidase/peroxidase (GOX/HRP) method (Anal. Biochem. 105 (1980), 389-397) specific activity.

The substrate solution was prepared by mixing 9 mL of soluble starch (1% in water, w/w) and 1 mL of 0.5 M sodium acetate buffer pH 5.0 in a 15-mL conical tube. A conjugated enzyme (GOX/HRP) solution with ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0) with final concentrations of 2.74 mg/mL ABTS, 0.1 U/mL HRP, and 1 U/mL GOX.

Serial dilutions of glucoamylase samples and glucose standards were prepared in purified water. Each glucoamylase sample (10 μL) was transferred to a new microtiter plate (Corning 3641) containing 90 μL of substrate solution pre-incubated at 50°C for 5 min at 600 rpm. The reaction was carried out at 50°C for 10 min (shaking (600 rpm) in a thermomixer (Eppendorf)), and 10 μL of the reaction mixture and 10 μL of serial dilutions of glucose standards were quickly transferred to new microtiter plates (Corning (Corning) Corning) 3641), then, 100 μL of ABTS/GOX/HRP solution was added. Absorbance at 405 nm was measured immediately using a SoftMax Pro microplate reader (Molecular Device) at 11 sec intervals for 5 min. The output is the reaction rate (Vo) for each enzyme concentration. Linear regression was used to determine the slope of the curve of Vo versus enzyme dose. Calculate the specific activity of glucoamylase based on a glucose standard curve using Equation 1:

Specific activity (unit/mg) = slope (enzyme) / slope (standard) × 1000 (1),

where 1 unit = 1 μmol glucose/min.

Using the method described above, the specific activity of PruGA1 was determined and compared to the benchmark AnGA (glucoamylase from Aspergillus niger). The results are shown in Table 2. The specific activity of PruGA1 for soluble starch was 197 U/mg, about 2 times higher than that of another glucoamylase, AnGA.

Table 2. Specific activity of purified PruGA1 on soluble starch compared to AnGA

Example 4

Amylopectin hydrolysis activity of glucoamylase PruGA1

Glucoamylase activity on pullulan was determined using the same protocol described above for the determination of specific activity of glucoamylase PruGA1 on soluble starch (except that the enzyme was dosed at 10 ppm). Table 3 summarizes the amylopectin hydrolysis activities of PruGA1 and benchmark AnGA. The activity of PruGA1 on amylopectin was approximately 6 times higher than that of AnGA on amylopectin.

Table 3. Amylopectin hydrolysis activity of PruGA1 compared to AnGA.

Example 5

Effects of pH and temperature on PruGA1 glucoamylase activity

The effect of pH (2.0 to 10.0) on PruGA1 activity was monitored using soluble starch (1% in water, w/w) as substrate. The buffered working solution consisted of a combination of glycine/sodium acetate/HEPES (250 mM), with pH varying from 2.0 to 10.0. Substrate solutions were prepared by mixing soluble starch (1% in water, w/w) with 250 mM buffer solution in a ratio of 9:1. Enzyme working solutions were prepared in water at a dose (showing signal in the linear range from the dose response curve). All incubations were performed at 50°C for 10 min following the same protocol as above for specific activity of glucoamylase PruGA1 on soluble starch. The enzymatic activity at each pH was reported as relative activity compared to the enzymatic activity at the optimum pH. The pH profile of PruGA1 is shown in Table 4. PruGA1 was found to have an optimum pH at about 5.0 and maintained more than 70% of its maximum activity between pH 2.8 and 7.0.

Table 4. pH profile of PruGA1

The effect of temperature (from 40°C to 84°C) on PruGA1 activity was monitored using soluble starch (1% in water, w/w) as substrate. Substrate solutions were prepared by mixing 9 mL of soluble starch (1% in water, w/w) and 1 mL of 0.5 M buffer (pH 5.0 sodium acetate) in a 15 mL conical tube. Enzyme working solutions were prepared in water at a dose (showing signal in the linear range from the dose response curve). Following the same protocol as above for the specific activity of glucoamylase PruGA1 on soluble starch, incubations were performed at temperatures ranging from 40°C to 84°C for 10 min, respectively. The activity at each temperature is reported as the relative activity compared to the enzyme activity at the optimum temperature. The temperature profile of PruGA1 is shown in Table 5. PruGA1 exhibited the best activity at 75°C and maintained more than 70% of the maximum activity between 63°C and 79°C.

Table 5. Temperature-activity curves of PruGA1

temperature(℃) Relative activity (%) 40 twenty four 44.7 34 49.4 42 55 54 59.7 63 65 77 69.2 90 74.6 100 80 58 85 19

Example 6

Saccharification performance of PruGA1 at pH 4.5 at different temperatures

The activities of PruGA1, AnGA and AfuGA (described in WO2014092960) were evaluated under saccharification conditions at pH 4.5 with different incubation temperatures. The assessment of DP1 was measured by analyzing sugar compositions with equal enzyme doses. Alpha-amylase pretreated corn starch liquefaction (prepared at 34.9% ds, pH 3.8) was used as the starting substrate. Incubations of glucoamylase (administered as 1× dose at 0.121 mg/gds) and corn starch liquefaction (34% ds) were performed at pH 4.5 at 60°C, 65°C and 70°C, respectively. Samples were collected at 16, 24, 40, 64 and 72 h, respectively. All incubations were quenched by heating at 100°C for 15 min. The sample supernatants were transferred and diluted 400-fold in ₅ mM _H2SO4 , using an Agilent 1200 series system (with Fast Fruit columns (100 mm x 7.8 mm)) running at 85°C for HPLC analysis. 10 μL of sample was loaded onto the column and separated using isocratic purified water as mobile phase at a flow rate of 1.0 mL/min. Oligosaccharide products were detected using a refractive index detector. The glucogenic activity of the samples is summarized in Table 6. Taking the DP1% after 72-h incubation (Fig. 3) as an example, the 0.3× dose (40 μg/gds) of PruGA1 was superior to AfuGA-1× dose (121 μg/gds) and AnGA-1 at all three tested temperatures x dose (121 μg/gds). Even when the incubation temperature was raised to 70 °C, PruGA1 maintained its excellent performance in DP1 production. The saccharogenic activities of the samples are summarized in Table 6.

Table 6. DP1 production of PruGA1, AfuGA and AnGA (1x dose set at 121 μg/gds) incubated with cornstarch liquefaction at pH 4.5 at different temperatures.

Example 7

PruGA1 glycation assessment at pH 5.5 and 70°C

The saccharogenic activity of PruGA1 was assessed at elevated temperature (in order to shorten the saccharification time). Corn starch liquefaction (32% ds, pH 3.9) was obtained from alpha-amylase pretreated corn starch liquefaction. Incubations with different doses of PruGA1 and cornstarch liquefaction (32% ds) were performed at pH 5.5, 70°C. Samples were collected at 18, 26, 42, 50, 66 and 72 h. All incubations were quenched by heating at 100°C for 15 min. The supernatants of the samples were transferred and diluted 400-fold in ₅ mM _H2SO4 for HPLC analysis using the same conditions as shown in Example 6. The saccharogenic activities of the samples are summarized in Table 7. The results showed that PruGA1 (administered at 30 μg/gds) could achieve >95% DP1 production after two days of incubation at pH 5.5 at 70°C.

Table 7. Using cornstarch liquefaction as substrate under saccharification conditions at pH 5.5 and 70°C,

Sugar production at different doses of PruGA1 (20 to 50 μg/gds)

Example 8

Crude starch activity of PruGA1

The activity of PruGA1 on crude starch was measured and compared to that of Trichoderma reesei glucoamylase (TrGA) for granular starch hydrolase (GSHE) fermentation and direct starch hydrolysis to glucose/maltose processes (DSTG/DSTM). In this assay, alpha-amylase and glucoamylase were mixed in a ratio of 1:6.6. Aspergillus kawachii amylase (AkAA, described in WO 2013169645) was used. Glycan profiling was performed using a Fast Fruit HPLC column (Waters) and the enzymatic crude starch hydrolysis capacity was determined using glucose (final product).

150 [mu]L of cornstarch substrate (1% in 50 mM pH 3.5/pH 4.5 acetate buffer) was dispensed into a 0.5 mL microtiter plate using wide-mouthed tips. 10 μL of amylase and 10 μL of glucoamylase were added per well to set the final doses of AkAA and glucoamylase to 1.5 ppm and 10 ppm, respectively. The samples were incubated in an iEMS incubator set at 32°C, 900 rpm for 6, 20 and 28 h. 50 μL of 0.5M NaOH was added to quench the reaction and the starch plug was suspended by placing the plate on a shaker for 2 min. Afterwards, the plate was sealed and centrifuged at 2500 rpm for 3 min. For HPLC analysis, the supernatant was diluted 10-fold with 0.01 NH ₂ SO ₄ . A 10 μL sample was analyzed using an Agilent 1200 series HPLC equipped with a refractive index detector. The column used was a Phenomenex Rezex-RFQFast Fruit column (Cat. No. 00D-0223-K0) with a Phenomenex Rezex ROA organic acid guard column (Cat. No. 03B-0138-K0). The mobile phase was 0.01 NH ₂ SO ₄ and the flow rate was 1.0 mL/min at 85°C. The results are shown in Figures 4 and 5. PruGA1 exhibited comparable activity to the benchmark glucoamylase TrGA on crude starch at both pH 3.5 and pH 4.5.

Example 9

Evaluation of PruGA1 in low pH fermentation

The glycogenic activity of PruGA1 was assessed under low pH fermentation conditions. The performance of PruGA1 and TrGA was tested at the same protein concentration (0.25 mg/gds). Amylase-treated corn starch liquefaction (34.9% ds, pH 3.8) was used as substrate. The pH of the cornstarch liquefaction (32% ds, amylase pretreated) was adjusted to pH 3.0 and 10 g were transferred to a 50 mL glass bottle. Incubations were performed at 32°C and 55°C. Samples were collected at 17, 24, 41, 48, 63, 72 h. All incubations were quenched by heating at 100°C for 15 min. The supernatants of the samples were transferred and diluted 400-fold in 5 mM H ₂ SO ₄ for HPLC analysis using the same conditions as shown in Example 7.

The values reported in Table 7 reflect the percent peak area for each DPn as a fraction of the total DP1, DP2, DP3 and DP3+. The data in Table 8 show that PruGA1 exhibited higher glycogenic activity than TrGA when fed at equal protein concentrations and incubated at 32°C, pH 3. When the incubation temperature was raised to 55 °C, PruGA1 hydrolyzed high DP sugars very efficiently, leaving only 5% DP3+ after 17 h incubation, while TrGA remained at 18%.

To further evaluate the performance of PruGA1 at low pH, another test was performed on starch liquefaction at an even lower pH (pH 2.0). The screening procedure was the same as at pH 3.0 (except that the enzyme was dosed at 0.2 mg/gds) and samples were collected at 4, 21, 29, 45, 53, 70 h. As shown in Table 9, at pH 2 and 32°C, PruGA1 released 77.4% of DP1 and TrGA released 54% after 70 h. The percentage of DP1 released by PruGA1 at 55 °C was 26.2%, while the TrGA response was only 3.2%.

Table 8. Sugar composition analysis of PruGA1 and TrGA hydrolyzed starch liquefaction at pH 3, 32°C or 55°C

Table 9. Sugar composition analysis of PruGA1 and TrGA hydrolyzed starch liquefaction at pH 2, 32°C or 55°C

Example 10

High temperature leaching saccharification with glucoamylase on wort substrate

The saccharogenic activity of PruGA1 during high temperature leaching saccharification was assessed compared to other glucoamylase benchmarks for wort substrates for brewing applications.

With 55% pilsner malt (Pilsner malt; Fuglsang, Denmark, batch 13.01.2016) and 45% corn flour (Benntag Nordic; Nordgetreide GmBH Lübec , Germany, Batch: 02.05.2016), using a 4.0:1 ratio of water to grain flour for the mashing operation. Pilsner malt was ground on a Buhler Miag malt grinder (0.5mm setting). Maize (1.35g), malt (ground pilsner malt, 1.65g) were mixed in a Wheaton cup (Wheaton glass vessel with lid), pre-incubated with 12.0g tap water, pH adjusted to pH 5.4 with 2.5M sulfuric acid . The resulting substrate (15% ds, pH 5.4) was then used for glucoamylase performance evaluation. PruGA1 (10 μL of a 1 mg/mL stock solution) was added to 90 μL of substrate dispensed in PCR microtiter plates (Axygen). Other glucoamylases evaluated were: TrGA variant A, Trichoderma reesei glucoamylase variant (with substitutions D44R and D539R, 10 μL of 2 mg/mL stock) and Aspergillus niger glucoamylase (AnGA, 10 μL of 1 mg/mL stock solution). All incubations were performed at 64°C for 4h or, for even higher saccharification temperatures, at 70°C for 2h; then at 79°C for 15min. After quenching the reaction at 95°C for 10 min, the reaction mixture was centrifuged at 3700 rpm for 10 min. Supernatant samples were transferred and diluted 20-fold in ₅ mM _H2SO4 for HPLC analysis. HPLC separation was performed using an Agilent 1200 series HPLC system at 85°C with a Fast fruit column (100 mm x 7.8 mm). The sample (10 μL) was loaded onto an HPLC column and separated using isocratic purified water as mobile phase at a flow rate of 1.0 mL/min. Oligosaccharide products were detected using a refractive index detector. The saccharogenic activities of the samples are summarized in Table 10. The 100 ppm PruGA1 sample exhibited comparable performance to the TrGA variant A (TrGA vA) glucoamylase at 200 ppm. The PruGA1 enzyme also showed better performance than the benchmark when the incubation temperature was increased to 70 °C and the incubation time was shortened to 2 h.

Table 10. Sugar composition analysis of glucoamylase incubated with wort substrate at pH 5.4

Example 11

High temperature leaching saccharification using malt and corn with glucoamylase

With 55% pilsner malt (Pilsner malt; Vogelsang, Denmark, batch 13.01.2016) and 45% corn flour (Nordic Bruntage; Nordgetreide GmBH Lübec, Germany, batch: 02.05.2016) The glucoamylase PruGA1 was tested in a mashing operation using a water to flour ratio of 4.0:1. Pilsner malt was ground using a Buhler Miag malt grinder (0.5mm setting).

Maize (1.35g) and malt (ground pilsner malt, 1.65g) were mixed in a Wheaton cup (Wheaton glass vessel with lid), pre-incubated with 12.0g tap water, pH adjusted to pH 5.4 with 2.5M sulfuric acid . Glucoamylase was added based on ppm active protein (1.0 ml total) and water was added as a no enzyme control. The Wheaton cup was placed in a Drybath (Thermo Scientific Stem station), magnetically stirred and the following saccharification procedure was applied; heat samples to 64°C for 30 minutes and hold at 64°C for 15 minutes; ℃/min increase temperature to heat to 79°C for 15 minutes; hold at 79°C for 15 minutes; heat to 90°C by 1°C/min increase temperature for 11 minutes, hold at 90°C for 15 minutes; cool Hold to 79°C for 15 minutes and finally heat to 79°C for 15 minutes and mashed off. Transfer 10 ml of sample to a Falcon tube and boil at 100°C for 20 minutes to ensure complete inactivation of the enzyme. At 10 The grains were separated from the wort by centrifugation at 4500 rpm in a Heraeus Multifuge X3R for 20 minutes at C. The supernatant was collected for HPLC sugar analysis using standard methods. The results are shown in Table 11.

Table 11. Relative distribution of sugars (DP1 to DP5+) in wort determined by HPLC

Clearly, PruGA1 promotes high production of DP1 in a dose-dependent manner. Up to 83.44% DP1 was produced at a dose of 750 ppm enzyme.

Example 12

100% corn saccharified with glucoamylase leaching at high temperature

The purpose of this experiment was to evaluate the saccharogenic activity of PruGA1 during high temperature mashing using corn and malt compared to industry benchmarks. The saccharification operation was carried out with 100% corn flour (Nordic Bruntage; Nordgetreide GmBH Lübec, Germany, batch: 02.05.2016) using a water to flour ratio of 4.0:1.

Raw corn (3.0 g) was added to a Wheaton cup (Wheaton glass vessel with lid), pre-incubated with 12.0 g tap water, and pH adjusted to pH 5.4 with 2.5M sulfuric acid. Glucoamylase was added based on ppm active protein (1.0 ml total), or water was added as a no enzyme control. A fixed concentration of 5.0 ppm of alpha-amylase (from DuPont) was applied in all samples 5T) and 0.21 ppm of beta-glucanase (from DuPont 750) to promote liquefaction and filterbility. A Wheaton cup was placed in the Drybath (Thermo Technologies main bench), magnetically stirred and three different mashing programs were applied. According to curve 1, the sample was heated to 64°C; held at 64°C for 80 minutes; heated to 80°C by increasing the temperature at 1.6°C/minute for 10 minutes; held at 80°C for 30 minutes, then washed out. According to curve 2, the sample was heated to 70°C; held at 70°C for 80 minutes; heated to 80°C for 10 minutes by increasing the temperature at 1.0°C/minute; held at 80°C for 30 minutes, then washed out; According to curve 3, the sample was heated to 75°C; held at 75°C for 80 minutes; heated to 80°C by increasing the temperature at 0.5°C/minute for 10 minutes; held at 80°C for 30 minutes, then washed out. Transfer 10 ml of sample to a Falcon tube and boil at 100°C for 20 minutes to ensure complete inactivation of the enzyme. The grains were separated from the wort by centrifugation in a Heraeus Multifuge X3R at 4500 rpm for 20 minutes at 10°C. The supernatant was collected for HPLC sugar analysis. The saccharogenic activities of the samples are summarized in Table 12.

Table 12. Sugar composition analysis of glucoamylase after leaching saccharification using 100% corn using various temperatures at pH 5.4.

Compared to TrGA (wild type from Trichoderma reesei glucoamylase) (final concentration: 18 ppm), PruGA1 showed enhanced performance (final concentration: 18 ppm) under the 70°C and 75°C saccharification profiles.

Claims (23)

1. A polypeptide having glucoamylase activity, selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity to the polypeptide of SEQ ID No. 3;

(b) a polypeptide encoded by a polynucleotide that hybridizes under at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency conditions, and even most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of seq id no

(i) The mature polypeptide coding sequence of SEQ ID NO. 1,

(ii) 1, or a genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO, or

(iii) (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, 97%, 98%, 99% or 100% identity to the mature polypeptide coding sequence of SEQ ID No. 1;

(d) a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., several) amino acids of the polypeptide of SEQ ID NO. 3; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that has glucoamylase activity.

2. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2 operably linked to one or more control sequences that control the production of the polypeptide in an expression host.

4. A recombinant host cell comprising the polynucleotide of claim 2.

5. The host cell of claim 4, which is an ethanologenic microorganism.

6. The host cell of claim 4 or 5, which further expresses and secretes one or more additional enzymes selected from the group consisting of proteases, hemicellulases, cellulases, peroxidases, lipolytic enzymes, metallolipolytic enzymes, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, alpha-amylases, pullulanases, phytases, tannases, pentosanases, maleases (malanases), beta-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, transferases, or combinations thereof.

7. A method for saccharifying starch-containing material, the method comprising the steps of: i) contacting the starch-containing material with an alpha-amylase; and ii) contacting the starch-containing material with a glucoamylase at a temperature of at least 70 ℃; wherein the process produces at least 70% free glucose from the starch-containing material (substrate).

8. The method of claim 7, wherein step (ii) is carried out at a temperature of at least 75 ℃, preferably at least 80 ℃, for between 12 and 96 hours, preferably for between 18 and 60 hours.

9. The process of claim 7 or 8, wherein the glucoamylase maintains at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% relative activity at a temperature of at least 70 ℃, and/or at a pH between 2.0 and 7.0, preferably between pH 4.0 and pH 6.0, more preferably between pH 4.5 and pH 5.5.

10. The method of any one of claims 7-9, wherein the method comprises performing step (i) and step (ii) sequentially or simultaneously.

11. The method of any one of claims 7-10, wherein the method further comprises a pre-saccharification prior to saccharification step ii).

12. The method of any one of claims 7-11, wherein the glucoamylase is the polypeptide of claim 1.

13. The method of any one of claims 7-12, wherein step (i) is carried out at or below the gelatinization temperature of the starch-containing material.

14. The method of any one of claims 7-13, wherein no additional debranching enzyme is present in step (i) and/or step (ii).

15. The method of claim 14, wherein the debranching enzyme is a pullulanase.

16. A saccharide produced by the method of any one of claims 7-15.

17. A method of producing a fermentation product from the sugar of claim 16, wherein the sugar is fermented by a fermenting organism.

18. The method of claim 17, wherein the fermentation process is carried out sequentially or simultaneously with step (ii).

19. The method of claim 17 or 18, wherein the fermentation product comprises ethanol.

20. The method of claim 17 or 18, wherein the fermentation product comprises a non-ethanol metabolite.

21. The method of claim 20, wherein the metabolite is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, organic acids, glucono delta-lactone, sodium erythorbate, omega 3 fatty acids, butanol, isobutanol, amino acids, lysine, tyrosine, threonine, glycinol, itaconic acid, 1, 3-propanediol, vitamins, enzymes, hormones, isoprene, or other biochemicals or biomaterials.

22. A method of using the polypeptide of claim 1 in brewing.

23. A method of using the polypeptide of claim 1 to produce beer or malt-based beverages.